Salidroside Reduces Cold-Induced Mucin Production by Inhibiting TRPM8 Activation

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 32: 637-646, 2013 Salidroside reduces cold-induced mucin production by inhibiting TRPM8 activation QI LI1, XIANG-DONG ZHOU1, VICTOR P. KOLOSOV2 and JULIY M. PERELMAN2 1Department of Respiratory Medicine, Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, P.R. China; 2Far Eastern Scientific Center of Physiology and Pathology of Respiration, Siberian Branch, Russian Academy of Medical Sciences, Blagoveschensk 675000, Russia Received April 18, 2013; Accepted June 28, 2013 DOI: 10.3892/ijmm.2013.1434 Abstract. Salidroside is an effective component of the tradi- gene expression in the absence of CREB activity. The results tional Chinese herb, Rhodiola rosea, that is known to have revealed that the cells treated with either CREB siRNA or sali- the ability to protect individuals from cold attacks. In the droside expressed low levels of TRPM8 mRNA and protein. present study, we investigated the effects of salidroside on These results indicate that salidroside reduces MUC overpro- respiratory epithelial cells exposed to cold temperatures. We duction induced by cold stimuli and that salidroside exerts its wished to determine whether salidroside exerts any effect protective effects by inhibiting TRPM8 activation, mainly by on cold-induced mucin (MUC) production and the possible decreasing CREB activity. mechanisms involved in this process. We incubated HBE16 cells with salidroside, exposed them to a cold stimulus (18˚C), Introduction and assayed the following endpoints: MUC production (the expression of MUC5AC), concentration intracellular of free Cold stimuli are known to exacerbate chronic obstructive calcium ([Ca2+]i), the activation of the transient receptor poten- pulmonary disease (COPD) and have been shown to promote tial melastatin 8 (TRPM8) channel and the cAMP response mucin (MUC) hypersecretion (1). Excessive MUC produc- element-binding protein (CREB). Our results revealed a tion leads to airway obstruction and enhances inflammation. significant increase in the [Ca2+]i concentration, as well as in Previous studies have mainly focused on secondary inflamma- TRPM8 and CREB expression in the cold-stimulated cells. tory processes (2) and bacterial (3) or viral infections (4,5) that MUC5AC expression was also increased. Treatment of the cells occur in the airways following exposure to cold temperatures; with salidroside at concentrations of 50 and 100 µM decreased however, the molecular mechanisms behind cold-induced the [Ca2+]i concentration, with a maximal effect detected in mucus secretion have not yet been fully elucidated. Human the cells treated with 100 µM salidroside. The expression of organs express ion channels of the transient receptor poten- TRPM8 and TRPM8 channel conductivity were also repressed tial (TRP) family, including some ion channels that respond by salidroside; salidroside decreased the high levels of CREB at distinct temperature thresholds (6-9). TRP melastatin 8 activity and phosphorylation observed in the cold-stimulated (TRPM8) is a member of this family that is activated by cells. Furthermore, we transfected the cold-stimulated cells temperatures ranging from 8 to 25˚C. TRPM8 is a ligand‑gated with CREB small interfering RNA (siRNA) to analyze TRPM8 cation channel with moderate to high selectivity for calcium ions (10,11). TRPM8 can be activated by cold temperatures and by cooling agents, such as menthol, eucalyptol and icilin. In Correspondence to: Professor Xiang-Dong Zhou, Department a previous study, we found that TRPM8 plays a critical of Respiratory Medicine, Second Affiliated Hospital, Chongqing role in cold-induced MUC secretion in the human respira- Medical University, No. 74 Linjiang Road, Chongqing 400010, tory system (12). Human airway epithelial cells exposed to P. R. Ch i na cold temperatures exhibited activated TRPM8 channels, E-mail: [email protected] and MUC5AC expression was upregulated through the TRPM8-Ca2+-phospholipase C (PLC)-phosphatidylinositol Abbreviations: TRPM8, transient receptor potential melastatin 8; CRE, cAMP response element; ELISA, enzyme-linked immuno- bisphosphate (PIP2) pathway. Thus, we hypothesized that sorbent assay; HRP, horseradish peroxidase; MUC, mucin; siRNA, TRPM8 plays a role in cold-induced MUC secretion. In this small interfering RNA study, we aimed to discover methods of inhibiting cold-induced MUC hypersecretion. Key words: salidroside, mucin, airway, cold stimuli, transient Salidroside (rhodioside) is a glycoside compound receptor potential melastatin 8 primarily found in the plant, Rhodiola rosea, which grows in cold, high-latitude areas. It is well known that salidroside has pharmacological effects on many pathological conditions, including radiation poisoning (13), inflammation (14), fatigue, 638 LI et al: SALIDROSIDE INHIBITS TRPM8 ACTIVATION aging and hypoxia (15,16). Other studies have found that siRNA transfection. Cells were plated at a density of salidroside exerts its protective effects mainly by repressing 1-2x106 cells/ml and incubated at 37˚C until the cells were the inflow of intracellular free calcium (Ca2+) ([Ca2+]i) into 60-80% confluent. CREB siRNA (0.5 µg) were diluted in cells. Considering what is already known about salidroside 100 µl of siRNA transfection medium (solution A); 6 µl and TRPM8, we hypothesized that salidroside has the ability siRNA transfection reagent was diluted in 100 µl of siRNA to protect cells from cold nociceptive stimuli and that it may transfection medium (solution B). The solutions (A + B) were play a positive role in the airway system. In the present study, subsequently mixed gently and incubated at room temperature we explored the regulation of TRPM8 gene transcription in for 45 min. The cells were washed once with 2 ml of siRNA cold-assaulted cells and examined the effects of salidroside on transfection medium followed by the addition of 0.8 ml of the TRPM8 channels and MUC production. We used the HBE16 siRNA transfection reagent mixture (solutions A + B). The human airway epithelial cell line for in vitro studies. The cells were then incubated for 7 h at 37˚C in a CO2 incubator cells were pre-treated with salidroside and exposed to cold and were recovered in 2 ml of DMEM/Ham's F12 medium temperatures; the protein and mRNA levels of MUC5AC were containing 10% FBS for an additional 24 h. The medium was then determined. The expression levels of TRPM8 and the then aspirated and replaced with fresh growth medium for the transcription factor, cAMP response element-binding protein following assays (see below for details). (CREB), were assayed to investigate whether there were any differences in the levels of these factors following treatment RNA isolation and real-time PCR for MUC5AC and TRPM8 with salidroside. Furthermore, we transfected the cells with mRNA. After the HBE16 cells were harvested, total RNA was CREB small interfering RNA (siRNA) to determine whether extracted from the cells using the Bioteke high-purity total the transcription factor, CREB, is involved in TRPM8 gene RNA extraction kit, and RNA integrity was verified using transcription. 1.5% agarose gel electrophoresis. The absorbance at 260/280 nm (A260/280) was in the range of 1.8-2.0. Total RNA was primed Materials and methods with an oligo(dT) primer and reverse transcribed using the PrimeScript RT Reagent kit. Real-time PCR was performed Reagents. Dulbecco's modified Eagle's medium (DMEM)/ using the SYBR Premix EX Taq™ II real‑time PCR kit. The Ham's F12 medium, HEPES, fetal bovine serum (FBS), sequences of the primers used for real‑time PCR were as anti-β-actin monoclonal antibody, salidroside (>98% purity; follows: MUC5AC (U06711) forward, 5'-CAGCCACGTCCCC National Institute for Food and Drug Control), CREB siRNA and TTCAATA-3' and reverse, 5'-ACCGCATTTGGGCATCC-3'; negative control siRNA were purchased from Sigma-Aldrich TRPM8 (NM024080) forward, 5'-ACTCAGAAGGCTGAGG (St. Louis, MO, USA). Lipofectamine® 2000 and OPTI-MEM TACA-3' and reverse, 5'-TTCAGTCGGAGTCTCACTCT-3'; Reduced Serum Medium were purchased from Invitrogen and GAPDH (BC026907) forward, 5'-GAAGGTGAAGGT (San Diego, CA, USA). The rabbit anti-TRPM8 polyclonal CGGAGT-3' and reverse, 5'-GAAGATGGTGATGGGA antibody was purchased from Abcam (Cambridge, MA, USA) TTTC-3'. GAPDH was used as the loading control. Relative and the horseradish peroxidase (HRP)-goat anti-rabbit IgG mRNA expression was determined by comparing it to a antibody was purchased from Santa Cruz Biotechnology, standard curve. Inc. (Santa Cruz, CA, USA). CREB-Luc reporter constructs were purchased from Biocat (Catalonia, Spain). The Dual Enzyme-linked immunosorbent assay (ELISA) for MUC5AC Luciferase Reporter Gene Assay kit was purchased from protein. MUC5AC protein levels in the cell lysates and BioTime Technology (Beijing, China). The pRL-TK Renilla supernatants were measured using ELISA. Cell lysates at Luciferase Reporter Vector was purchased from Promega multiple dilutions were prepared with phosphate-buffered (Madison, WI, USA). The high-purity total RNA extraction kit saline (PBS). Cell culture supernatants were collected for was purchased from Bioteke Biotechnology (Beijing, China). this assay. In addition, 50 µl of each sample were incubated The PrimeScript RT Reagent kit was purchased from Takara with bicarbonate‑carbonate buffer (50 µl) at 40˚C in a 96‑well (Dalian, China). plate until dry. The wells were washed 3 times with PBS and blocked with 2% bovine serum albumin (BSA) for 1 h at room Cell culture. HBE16 human airway epithelial

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